Chromatophores and color change in the lizard,Anolis carolinensis

Summary The skin of the lizard, Anolis carolinensis, changes rapidly from bright green to a dark brown color in response to melanophore stimulating hormone (MSH). Chromatophores responsible for color changes of the skin are xanthophores which lie just beneath the basal lamina containing pterinosomes and carotenoid vesicles. Iridophores lying immediately below the xanthophores contain regularly arranged rows of reflecting platelets. Melanophores containing melanosomes are present immediately below the iridophores. The ultrastructural features of these chromatophores and their pigmentary organelles are described. The color of Anolis skin is determined by the position of the melanosomes within the melanophores which is regulated by MSH and other hormones such as norepinephrine. Skins are green when melanosomes are located in a perinuclear position within melanophores. In response to MSH, they migrate into the terminal processes of the melanophores which overlie the xanthophores above, thus effectively preventing light penetration to the iridophores below, resulting in skins becoming brown. The structural and functional characteristics of Anolis chromatophores are compared to the dermal chromatophore unit of the frog.This study was supported in part by GB-8347 from the National Science Foundation.Contribution No. 244, Department of Biology, Wayne State University.The authors are indebted to Dr. Joseph T. Bagnara for his encouragement during the study and to Dr. Wayne Ferris for his advice and the use of his electron microscope laboratory.     

An Ultrastructural and Carotenoid Analysis of the Red Ventrum of the Japanese Newt,Cynops pyrrhogaster

The ventral skin of the wild Japanese newt Cynops pyrrhogaster is creamy at metamorphosis, but turns red when mature. The color of the ventral skin of laboratory (lab)‐reared newts stays yellow throughout their life. However, the mechanism for the red coloration of this animal still remains unknown. In this study, we have performed ultrastructural and carotenoid analyses of the red ventrum of wild and lab‐reared Japanese newts. Using electron microscopy, we observed a number of xanthophores having ring carotenoid vesicles (rcv) and homogenous carotenoid granules (hcg) in the ventral red skin of the wild newt. In the skin, β‐carotene and five other kinds of carotenoids were detected by thin‐layer chromatography (TLC). In the ventral yellow skin of lab‐reared newts, however, only β‐carotene and three other kinds of carotenoids were found. The total amount of carotenoids in the red skin of the wild adult newt was six times more than that of the yellow skin of the lab‐reared newt. Moreover, rcv were more abundant in xanthophores in red skin, but hcg were more abundant in yellow skin. These results, taken together, suggest that the presence of carotenoids in rcv in xanthophores is one of the critical factors for producing the red ventral coloration of the Japanese newt C. pyrrhogaster.     

Ultrastructural changes in the dermal chromatophore unit of Hyla arborea during color change

Summary The structural changes in the chromatophores of Hyla arborea related to changes in skin color were studied by electron microscopy and reflectance microspectrophotometry. During a change from a light to a darker green color, the melanosomes of the melanophores disperse and finally surround the iridophores and partly the xanthophores. The iridophores change from cup-shape to a cylindrical or conical shape with a simultaneous change in the orientation of the platelets from being parallel to the upper surface of the iridophores to being more irregular. The xanthophores change from lens-shape to plate-shape. The color change from green to grey seems always to go through a transitional black-green or dark olive green to dark grey. During this change the xanthophores migrate down between the iridophores, and in grey skins they are sometimes found beneath them. The pterinosomes gather in the periphery of the cell, while the carotenoid vesicles aggregate around the nucleus. The iridophores in grey skin are almost ball-shaped with concentric layers of platelets. A lighter grey color arises from a darker grey by an aggregation of melanosomes. The chromatophore values previously defined for Hyla cinerea are applicable in Hyla arborea, and the ultrastructural studies support the assumptions previously made to explain these values.The author wishes to thank Drs. P. Budtz, J. Dyck and L.O. Larsen for valuable discussions and J. Dyck for kindly providing the spectrophotometer granted him by the Danish National Science Foundation. The skilled technical assistance of Mrs. E. Schiøtt Hansen is gratefully acknowledged. Permission was granted by the Springer-Verlag to republish the illustrations of W.J. Schmidt (1920)     

Pigment cell differentiation in the fire-bellied toad,Bombina orientalis. I. Structural,chemical, and physical aspects of the adult pigment pattern

Wild-collected adults of Bombina orientalis are bright green dorsally and red to red-orange ventrally. As a prelude to an analysis of the differentiation of pigment cells in developing B. orientalis, we describe structural and chemical aspects of the fully differentiated pigment pattern of the “normal” adult. Structurally, differences between dorsal green and ventral red skin are summarized as follows: (1) Dorsal green skin contains a “typical” dermal chromatophore unit comprised of melanophores, iridophores, and xanthophores. Red skin contains predominantly carotenoid-containing xanthophores (erythrophores), and skin from black spot areas contains only melanophores. (2) In ventral red skin, there is also a thin layer of deep-lying iridophores that presumably are not involved in the observed color pattern. (3) Xanthophores of red and green skin are morphologically distinguishable from each other. Dorsal skin xanthophores contain both pterinosomes and carotenoid vesicles; ventral skin xanthophores contain only carotenoid vesicles. Carotenoid vesicles in dorsal xanthophores are much larger but less electron dense than comparable structures in ventral xanthophores. The presence of carotenes in ventral skin accounts for the bright red-orange color of the belly of this frog. Similar pigments are also present in green skin, but in smaller quantities and in conjunction with both colored (yellow) and colorless pteridines. From spectral data obtained for xanthophore pigments and structural data obtained from the size and arrangement of reflecting platelets in the iridophore layer, we attempt to explain the phenomenon of observed green color in B. orientalis.     

The Erythrophore in the Larval and Adult Dorsal Skin of the Brown Frog,Rana ornativentris: Its Differentiation,Migration, and Pigmentary Organelle Formation

To determine whether or not the erythrophore originates from xanthophores in the dorsal skin of the brown frog, Rana ornativentris, we morphologically examined the differentiation and migration of the two chromatophore types and their pigmentary organelle formation. At an early tadpole stage, three kinds of chromatophores, xanthophores, iridophores, and melanophores, appeared in the subdermis, whereas the erythrophore did so just before the foreleg protrusion stage. By the middle of metamorphosis, most chromatophores other than erythrophores had migrated to the subepidermal space. Erythrophores, which appeared late in the subdermis, proliferated actively there during metamorphosis and finished moving into the subepidermal space by the completion of metamorphosis. Carotenoid vesicles and pterinosomes within the erythrophores and xanthophores showed several significant differences in structure. In xanthophores, carotenoid vesicles were abundant throughout life, whereas those in erythrophores decreased in number with the growth of the frogs. The fibrous materials contained in the pterinosomes were initially scattered but soon formed a concentric lamellar structure. In erythrophores, the lamellar structure began to form at the periphery of the organelles but at the center in xanthophores. In addition, the pterinosomes of erythrophores were uniform in size throughout development, while those of xanthophores showed a tendency to become smaller after metamorphosis. The pterinosomes of xanthophores were significantly larger than those of erythrophores. These findings suggest that an erythrophore is not a transformed xanthophore, although they resemble each other closely in many respects.     

Changes in the distribution of melanophores and xanthophores inTriturus alpestris embryos during their transition from the uniform to banded pattern

Summary The change in distribution of melanophores from stage 28+ (uniform melanophore pattern) to stage 34 (banded melanophore pattern) and the participation of xanthophores in these changes has been investigated inTriturus alpestris embryos by studying the social behaviour of single cells. While melanophores are clearly visible from outside the embryo at stage 28+, xanthophores cannot be recognized from the outside until after stage 34. In ultrathin sections of stage 34 embryos, xanthophores are observed alternating with melanophores in a zonal distribution (Epperlein 1982). Using detached pieces of dorsolateral trunk skin, which retain their chromatophores after detachment, the entire distribution of melanophores and xanthophores can be visualized in a scanning electron microscope (SEM). In ambiguous cases (early stages), cells were reprocessed for transmission electron microscopy (TEM) and the presence of the characteristic pigment organelles was assessed. In addition, xanthophores were specifically identified by pteridine fluorescence with dilute ammonia. Pteridines were also identified chromatographically in skin homogenates. The combination of these methods allowed precise identification and quantitative determination of melanophores and xanthophores. Both cell types were present as codistributed, biochemically differentiated cells at stage 28+. Changes in the pattern up to stage 34 were due to the rearrangement at the epidermal-mesodermal interface of a relatively fixed number of melanophores which became preferentially localised at the dorsal somite edge and at the lateral plate mesoderm, and to the distribution of an increasing number of xanthophores to subepidermal locations in the dorsal fin and between the melanophore bands. Concomitant was an increase in the thickness of the epidermal basement membrane and a change in shape of chromatophores from elongate via stellate to rosette shaped, which may be correlated with a shift from migratory to sessile phases.     

Ultrastructural Immunogold Localization of Some Organelle-Transport Relevant Proteins in Wholemounted Permeabilized Nonextracted Goldfish Xanthophores

By whole-cell transmission electron microscopy (WCTEM), we recently demonstrated that carotenoid droplets are transported by elongating or retracting endoplasmic reticular cisternae in goldfish xanthophores. Here we report that permeabilized xanthophores demonstrate immunogold reactivity against several proteins involved in organelle translocation. The gold labeling against β-tubulin and the intermediate filament protein p45a were found on microtubules and intermediate filaments. Labeling with antiactin was found on nonidentifiable structures, on vesicles of unknown origin, occasional labeling on carotenoid droplets, and on occasional microfilaments. Immunoreactivity was demonstrated with anti-p57 on the carotenoid droplet surface, confirming previous results (Lynch et al., 1986a,b). Labeling with anti-PCD6 subunit (of the inositol trisphosphate/ryanodine receptor) was demonstrated on carotenoid droplets suggesting they possess calcium channels. Anti-MAP 1C (dynein) immunolabeling was generally seen on club-shaped structures in the cytomatrix and on carotenoid droplets. Finally, immunogold labeling with anti-MAP 2a + 2b was seen on a meshwork of microfilaments and intermediate filaments. Finally, this is the first report of a WCTEM technique for permeabilized cells that reveals immunoreactive elements, organelles, and cytomatrix components without the additional requirements of extraction or fracturing.     

An ultrastructural and carotenoid analysis of the red ventrum of the Japanese newt,Cynops pyrrhogaster

The ventral skin of the wild Japanese newt Cynops pyrrhogaster is creamy at metamorphosis, but turns red when mature. The color of the ventral skin of laboratory (lab)-reared newts stays yellow throughout their life. However, the mechanism for the red coloration of this animal still remains unknown. In this study, we have performed ultrastructural and carotenoid analyses of the red ventrum of wild and lab-reared Japanese newts. Using electron microscopy, we observed a number of xanthophores having ring carotenoid vesicles (rcv) and homogenous carotenoid granules (hcg) in the ventral red skin of the wild newt. In the skin, beta-carotene and five other kinds of carotenoids were detected by thin-layer chromatography (TLC). In the ventral yellow skin of lab-reared newts, however, only beta-carotene and three other kinds of carotenoids were found. The total amount of carotenoids in the red skin of the wild adult newt was six times more than that of the yellow skin of the lab-reared newt. Moreover, rcv were more abundant in xanthophores in red skin, but hcg were more abundant in yellow skin. These results, taken together, suggest that the presence of carotenoids in rcv in xanthophores is one of the critical factors for producing the red ventral coloration of the Japanese newt C. pyrrhogaster.     

Dermal and epidermal chromatophores of the Antarctic teleost Trematomus bernacchii

The physiological response and ultrastructure of the pigment cells of Trematomus bernacchii, an Antarctic teleost that lives under the sea ice north of the Ross Ice Shelf, were studied. In the integument, two types of epidermal chromatophores, melanophores and xanthophores, were found; in the dermis, typically three types of chromatophores--melanophores, xanthophores, and iridophores--were observed. The occurrence of epidermal xanthophore is reported for the first time in fish. Dermal melanophores and xanthophores have well-developed arrays of cytoplasmic microtubules. They responded rapidly to epinephrine and teleost melanin-concentrating hormone (MCH) with pigment aggregation and to theophylline with pigment dispersion. Total darkness elicited pigment aggregation in the majority of dermal xanthophores of isolated scales, whereas melanophores remained dispersed under both light and dark conditions. Pigment organelles of epidermal and dermal xanthophores that translocate during the pigmentary responses are carotenoid droplets of relatively large size. Dermal iridophores containing large reflecting platelets appeared to be immobile.     

Actin-dependent carotenoid droplet dispersion in permeabilized cultured goldfish xanthophores

Organelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule-dependent organelle translocations, little is known about actin-dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actin-dependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin-permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti-actin, but not by anti-tubulin or anti-intermediate filament proteins, suggesting a dependence on F-actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.     

Ultrastructural changes in suspension culture cells of Panicum maximum during cryopreservation

A Panicum maximum cell suspension was used to study ultrastructural changes during cryopreservation. Pregrowing the cells in mannitol caused reduction in the vacuolar volume by redistribution of the large central vacuole into a number of smaller vesicles. Invaginations were formed in the plasma membrane of the cells, to accommodate the reduced cell volume. Swelling of organelles occurred during different stages of cryopreservation. The cisternae of the endoplasmic reticulum dilated and formed vesicles. Although some damage was apparent, organelles were still recognizable in cells frozen slowly and freeze-fixed at –10°C. The cells were able to repair such damage within two days in culture, and regained their normal appearance. Cells frozen slowly without any cryoprotection, and cells frozen rapidly by direct immersion into liquid nitrogen after cryoprotection, were lethally damaged by destruction of membranous structures. Osmiophilic granules were found along the plasma membrane of lethally damaged cells, indicating that their formation is a consequence of freeze damage, rather than a mechanism to prevent injury.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide     

Cytochemical localization of cellulase activity in pollen mother cells of david lily during meiotic prophase I and its relation to secondary formation of plasmodesmata

Summary Cellulase activity was localized at the ultrastructural level in pollen mother cells (PMCs) of David lily [Lilium davidii var.willmottiae (Wilson) Roffill] at different stages of meiotic prophase I. The enzyme was observed to appear at the early leptotene stage and reached its highest level at the subsequent zygotene stage, and its subcellular distribution revealed by the presence of electron-dense deposits of reaction product was found to be restricted exclusively to the endoplasmic reticulum (ER), the vesicles derived from that, and the cell wall, especially at the sites of secondary plasmodesmata and cytoplasmic channels where the wall was being digested. Other cytoplasmic organelles, such as dictyosomes and Golgi vesicles, lacked such deposits of reaction product. After zygotene the enzyme activity decreased abruptly, and at the pachytene stage only very few deposits could be observed in the cell wall. Our results indicate that cellulase is synthesized on rough ER and secreted directly via the smooth ER and ER-derived vesicles into the cell wall by exocytosis, where it brings about local wall breakdown, leading to the secondary formation of plasmodesmata and cytoplasmic channels.     

Xanthophores in chromatophore groups of the premigratory neural crest initiate the pigment pattern of the axolotl larva

Summary The barred pigment pattern (Lehman 1957) of the axolotl larva is best observed from stage 41 onwards, where it already consists of alternating transverse bands of melanophores and xanthophores along the dorsal side of the trunk. The present study investigateswhen the two populations of neural crest derived chromatophores, melanophores and xanthophores become determined andhow they interact to create the barred pigment pattern. The presence of phenol oxidase (tyrosinase) in melanophores (revealed by dopa incubation) and pteridines in xanthophores (visualized by fluorescence) were used as markers for cell differentiation in order to recognize melanophores and xanthophores before they became externally visible. It was found that melanophores and xanthophores were already determined in the premigratory neural crest, at stages 30/31 and 35–36, respectively. Between stages 35–36 and 38 they were arranged in a prepattern of several distinct, mixed chromatophore groups along the dorsal trunk, morphologically correlated in the scanning electron microscope with humps on the original crest cell string. While the occurrence of xanthophores was restricted to the chromatophore groups and around them, melanophores were already uniformly distributed in the dorsolateral flank area, having migrated from trunk neural crest portions including the groups. The bar component of the pigment pattern was subsequently initiated by xanthophores, which caused melanophores in and around the chromatophore groups to fade or become invisible. The barred pattern was established by the formation of alternating clusters of like cells, melanophores and xanthophores.     

Different distribution of melanophores and xanthophores in early tailbud and larval stages inTriturus alpestris

Summary The subepidermal distribution of xanthophores and melanophores is investigated in embryos ofTriturus alpestris with a uniform (stage 28+) and a banded melanophore pattern (stage 35/36). In ultrathin head and trunk sections from stage 35/36 embryos which externally show longitudinal dorsal and lateral melanophore bands in the trunk and less compact continuations of the dorsal bands in the head, xanthophores were discovered in addition to melanophores. Melanophores contain melanosomes while xanthophores which are not externally visible, are recognized by their pterinosomes. Both chromatophore cell types are mutually exclusively distributed on the epidermal basement membrane (bm). Mesenchymal cells seemed not to be able to replace them, except on the bm of the corneal epithelium where there were only mesenchymal cells. In head and trunk sections from stage 28+ embryos which externally show a distribution of uniformly scattered melanophores on the dorsolateral halves, melanophores were found on the dorsolateral neural crest migration route. No epidermal bm was present and xanthophores were undetectable. In ventrolateral and ventral portions of embryos of both stages no chromatophores occurred. This investigation defines the histological localization of melanophores and xanthophores in embryos with a typical uniform and banded melanophore arrangement; a subsequent study analyzes when xanthophores appear and how they arrange with melanophores in alternating zones.     

Ultrastructural demonstration of hormone-induced movement of carotenoid droplets and endoplasmic reticulum in xanthophores of the goldfish,Carassius auratus L.

Summary The hormone-induced pigment dispersion in primary cultures of xanthophores of goldfish (Carassius auratus L.) has been shown to involve the dispersion of not only carotenoid droplets but also of smooth endoplasmic reticulum. The dispersion of these organelles is inhibited by cytochalasin B and is accompanied by thinning of the cell body, thickening of the processes, and also overall changes in cellular morphology (process extension) under certain conditions. Electron microscopic examination of heavy meromyosin treated glycerinated xanthophores in scales revealed the presence of actin filaments in these cells.This work was supported, in part, by grants AM-5384 and AM-13724 from U.S.P.H.S.     

Identification of pigment cells during early amphibian development (Triturus alpestris,Ambystoma mexicanum)

Summary The purpose of the present investigation was to provide and apply a methodological manual with which the distribution, patterning and relationship of melanophores and xanthophores can be analyzed during early amphibian development. For demonstration of the methods, which include ultrastructural, histochemical and biochemical approaches, Triturus alpestris and Ambystoma mexicanum (axolotl) embryos are used. These two species differ conspicuously in their larval pigment patterns, showing alternating melanophore bands in horizontal (T. alpestris) and vertical (axolotl) arrangements. With transmission- and scanning electron microscopy melanophores and xanthophores were distinguished by their different pigment organelles and surface structures. The presence of phenol oxidase (tyrosinase) was used to reveal externally invisible or faintly visible melanophores by applying an excess of 3,4 dihydroxy-phenylalanine (dopa). Xanthophores were made visible in fixed and living embryos by demonstrating their pterin fluorescence. In addition, pterins were analyzed by HPLC in embryos before and after pigmentation was visible.Abbreviations DOPA dihydroxy-phenylalanine - FCS fetal calf serum - FIF formaldehyde-induced fluorescence - FITC fluorescein isothiocyanate - HPLC high performance liquid chromatography Dedicated to the memory of Dr. Michael Claviez     

Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes

In goldfish xanthophores, the formation of pigment aggregate requires: 1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; 2) that self-association of pigment organelles occur in a microtubule-independent manner; and 3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP-dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F-actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass approximately 72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5x lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.     

Rearrangements of pterinosomes and cytoskeleton accompanying pigment dispersion in goldfish xanthophores

The cytoskeleton of goldfish xanthophores contains an abundance of unique dense structures (400 nm in diameter) that are absent in goldfish nonpigment cells and are probably remnants of pterinosomes. No major difference in protein composition between xanthophores and nonpigment cells (without these structures) was found that could account for these structures. In xanthophores, these structures are foci of radiating filaments. The addition or withdrawal of ACTH causes a radical rearrangement of the xanthophore cytoskeleton accompanying redistribution of carotenoid droplets, namely, the virtual exclusion of these dense bodies with associated filaments from the space occupied by the carotenoid droplet aggregate vs. a relatively even cytoplasmic distribution of these structures when the carotenoid droplets are dispersed. These changes in cytoskeletal morphology are not accompanied by any major changes in the protein or phosphoprotein composition of the cytoskeleton.     

Ultrastructural changes in major organelles during spermatial differentiation inBangia (Rhodophyta)

Summary The major organelles within the cells of maleBangia atropurpurea (Roth) C. Ag. filaments undergo a series of ultrastructural transformations during the production of spermatia. Initially, thylakoids within the large axial chloroplast develop a reticulate pattern commencing at the central pyrenoid region. Subsequent changes involve loss of lobes and diminution of volume through division; chloroplasts in final stages contain a few dilated, distorted thylakoids and many plastoglobuli. During differentiation the large nucleolus disappears from the nucleus and four masses of chromatin aggregate near the nuclear envelope. Furrows originating from the nuclear envelope form double membranes around each of the chromatin masses and most of the nucleoplasm is eliminated. Several types of fibrillar vesicles are formed during the process and large floridean starch reserves are utilized. Multilamellar bodies and microbody-like structures occur within the cells during certain phases of spermatiogenesis.     

Ultrastructural observations of mature and encysting zoospores ofPythium proliferum de Bary

Summary The process of zoospore maturation and encystment inP. proliferum was studied by electron microscopy. General ultrastructural features of the mature, swimming zoospore were found to be similar to those previously described for other oomycetes in both the attachment and ultrastructure of the flagella as well as the type and distribution of cellular organelles. Associated with extensive areas of RER in the mature zoospores were unusual, electrondense, bar-like structures. These structures were found in the groove region of young zoospores and at the periphery of encysting zoospores. Their possible function is discussed. The five main types of vesicles observed during encystment, as seen grouped in this study, along with the vesicles described in previous studies of oomycete encystment, were in table form and individually discussed. Interesting correlations appear to exist in the types of vesicles that are present within the oomycetes studied thusfar.